Shirlei Maria Recco-Pimentel

Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura, Leptodactylidae) detected by silver staining and fluorescence in situ hybridization.

The nucleolus organizer regions (NORs) of both karyotypes I and II of Physalaemus petersi (Jiménez de la Espada, 1872) from the Brazilian Amazon were studied by Giemsa staining, and by the Ag- NOR method. Karyological group I specimens were also studied by the fluorescence in situ hybridization (FISH) technique. Multiple NOR-bearing chromosomes were detected in both karyotypes. The coincident results of the Ag-NOR and FISH methods rule out the occurrence of silent NORs in this anuran. There was no intraindividual NOR variability in either group, but interindividual variability of NORs was high in group I. Seven different patterns of active NOR distribution were definitely recognized among fifteen specimens. This was considered to be a NOR site polymorphism. These results, combined with the C-band polymorphism previously reported for P. petersi, demonstrate a high rate of chromosome evolution in this group.

The ultrastructure of the spermatozoa of Epipedobates flavopictus (Amphibia, Anura, Dendrobatidae), with comments on its evolutionary significance.

We describe, for the first time, the spermatozoon ultrastructure of a dendrobatid frog, Epipedobates flavopictus. Mature spermatozoa of E. flavopictus are filiform, with a moderately curved head and a proportionally short tail. The acrosomal vesicle is a conical structure that covers the nucleus for a considerable distance. A homogeneous subacrosomal cone lies between the acrosome vesicle and the nucleus. The nucleus contains a nuclear space at its anterior end, and electron-lucent spaces and inclusions. No perforatorium is present. In the midpiece, the proximal centriole is housed inside a deep nuclear fossa. Mitochondria are scattered around the posterior end of the nucleus and inside the undulating membrane in the anterior portion of the tail. In transverse section the tail is formed by an U-shaped axial fiber connected to the axoneme through an axial sheath, which supports the undulating membrane. The juxta-axonemal fiber is absent. The spermatozoon of E. flavopictus has several characteristics not observed before in any anurans, such as a curved axial fiber, absence of a juxta-axonemal fiber, and presence of mitochondria in the typical undulating membrane. Our results endorse the view that, in anurans, the conical perforatorium and subacrosomal cone are homologous and that Dendrobatidae should be grouped within Bufonoidea rather than Ranoidea.

Cytogenetics of three Brazilian species of Eleutherodactylus (Anura, Leptodactylidae) with 22 chromosomes and re-analysis of multiple translocations in E. binotatus

In this paper, we provide a cytogenetic analysis of Eleutherodactylus guentheri, E. parvus and E. binotatus. All of the species had a diploid chromosomal number of 2n = 22. The karyotypes of E. guentheri and E. parvus were very similar and differed only slightly in the morphology of pair 2. These two species also had an NOR-bearing secondary constriction on the long arms of pair 6. The karyotype of E. binotatus differed from those of E. guentheri and E. parvus in the morphology and size of the chromosomes, in the number of chromosomal arms, in the NOR location (detected on the short arms of pair 1), and in the pattern of heterochromatin. These results reinforce the differences between E. guentheri and E. binotatus and support the existence of two species group. Five individuals of E. binotatus showed morphs for pairs 2 and 3. These morphs probably arose from the translocation of a segment from one chromosome of pair 3 to a homologue of pair 2. In addition, some mitotic metaphases of E. binotatus showed spontaneous chromosomal breaks which suggested that there were sites of fragility. Meiotic diakinesis showed multiple chromosomal rings, indicating the occurrence of multiple translocations, as previously reported by other investigators. These data suggest that, in addition to fission and fusion, other chromosomal rearrangements were probably involved in the differentiation of the karyotypes of these species of Eleutherodactylus, especially E. binotatus.

Phylogenetic relationships of Pseudis and Lysapsus (Anura‚ Hylidae‚ Hylinae) inferred from mitochondrial and nuclear gene sequences

The previous uncertain placement of Lysapsus and Pseudis within the neobatrachians was recently resolved by molecular and morphological studies, which supported them as members of the Hylinae subfamily. Their inter‐ and intrageneric relationships, however, have long been under debate and no studies shed light on these questions. Aiming to elucidate such questions, this paper used 3.2 kb comprising the mitochondrial genes 12S, tRNA valine, 16S and cytochrome b, and the nuclear exon 1 coding for rhodopsin, to all representatives of both genera (except to two subspecies of Pseudis paradoxa). The results identified three major clades: the clade 1 was composed by Lysapsus species and subspecies; clade 2 included the subspecies of the Pseudis paradoxa (Pseudis paradoxa paradoxa, P. paradoxa platensis and P. paradoxa occidentalis), P. fusca, P. bolbodactyla and P. tocantins, and clade 3 was composed by Pseudis southern Brazil species (Pseudis cardosoi and P. minuta). As closely related taxa we found Pseudis minuta + P. cardosoi; P. tocantins + P. fusca, and the subspecies within each genus. Evidence that Pseudis is not monophyletic with respect to Lysapsus was found and we suggest Lysapsus to be a junior synonym of Pseudis. Based on pair‐wise comparison among gene sequences, we also suggest that the subspecies of Pseudis paradoxa and Lysapsus limellum must be considered as full species.

CYTOGENETIC ANALYSIS OF FOUR POISON FROGS OF THE EPIPEDOBATES GENUS (ANURA: DENDROBATIDAE)

We determined the overall karyotypic structure, positions of NORs, as well as the distribution of constitutive heterochromatin for Epipedobates flavopictus, E. trivittatus, E. femoralis, and E. hahneli. Despite a conserved chromosome number (2N = 24), morphological differences were seen in the group of small chromosomes. Epipedobates femoralis presented a distinctive karyotype compared to the other species analyzed. All species examined had NORs on different chromosomes. The C-banding pattern showed a considerable variation among the species. Epipedobates flavopictus is remarkably different from the others in possessing only centromeric C-bands. The distribution of heterochromatin varied among species and seems to evolve as a species-specific trait. We suggest that NOR location variability indicates that some rearrangement mechanisms have taken place during the evolutionary history of this group, because Dendrobatidae is considered a monophyletic taxon. We do not discard that some general pattern in either C-band or NOR location, or both, may emerge when more species of this genus are analyzed.

Chromosomal rearrangements as the source of variation in the number of chromosomes in Pseudis (Amphibia, Anura)

A cytogenetic study of Pseudis specimens from three localities in Rio Grande do Sul State, the southernmost Brazilian, was performed to identify karyotypic characteristics that could account for differences in vocalization pattern and body size. Individuals from around Tainhas were compared to those of São Jerônimo and Eldorado do Sul. Specimens from these latter two localities were identified as Pseudis minuta, while those from the former were classified as Pseudis sp. (aff. minuta). The populations from São Jerônimo and Eldorado do Sul had 2n = 24 chromosomes, classified as metacentric, submetacentric and subtelocentric. The population from Tainhas had 2n = 28 chromosomes, with four pairs of telocentric chromosomes. Modelling of these 28 chromosomes and testing for fusion in the centromeric/telomeric regions yielded a karyotype of 2n = 24 chromosomes, similar to that of the other populations. The similarity was reinforced by the location of the NORs and heterochromatin. The Tainhas population showed an increase in heterochromatin, as seen by the presence of additional C-bands, especially in the telocentric chromosomes. These data suggest that the two karyotypes described in this work had a common ancestry. There is evidence that the differentiation of these karyotypes may have occurred by chromosome fission and heterochromatin addition. Based on the present karyotype (2n = 28) and on morphological and vocalization studies by other researchers, we conclude that the Tainhas population may represent a new species.

NOR Dispersion, Telomeric Sequence Detection in Centromeric Regions and Meiotic Multivalent Configurations in Species of the Aplastodiscus albofrenatus Group (Anura, Hylidae)

In the present study, we examined the chromosome number and morphology, heterochromatin pattern, and location of the nucleolar organizer region (NOR) and telomeric sequences in Aplastodiscus albofrenatus, A. arildae, A. ehrhardti and A. eugenioi in order to cytogenetically characterize the Aplastodiscus albofrenatus group. The 4 species analyzed in this study had diploid numbers of 2n = 22 and very similar chromosome morphology. These species could be differentiated based on the distribution and amount of heterochromatin and the location of the nucleolar organizer region (NOR). Six of the 8 A. albofrenatus individuals had an NOR polymorphism previously unknown in anurans since only one of the homologs of pairs 1 and 7 was stained. In the other 2 specimens, the NOR occurred on both homologs of pair 7. In A. ehrhardti, pairs 6 and 10 were stained by the AgNOR technique, but only pair 6 was confirmed by in situ hybridization. The NOR was located on pair 10 in A. arildae and on pair 7 in A. eugenioi. In A. albofrenatus and A. arildae, multivalent rings involving NOR-containing chromosomes were observed during prophase I of meiosis. The telomeric probe identified the telomeres in all species and also centromeric regions in the chromosomes of A. albofrenatus and A. arildae, which were coincident with centromeric heterochromatin. The conserved chromosomal morphology seen mainly in the first 7 pairs among species of the A. albofrenatus group suggests that all of these species probably originated from a common ancestral karyotype and that chromosomal rearrangements resulted in karyotype differentiation, with changes in NOR location, as well as telomeric and heterochromatin dispersal.

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.

Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences.

Although some species groups have been recognized in the leiuperine genus Physalaemus, no phylogenetic analysis has previously been performed. Here, we provide a phylogenetic study based on mitochondrial and nuclear DNA sequences from 41 of the 46 species of Physalaemus. We employed the parsimony criterion using the software TNT and POY and the Bayesian criterion using the software MrBayes. Two major clades were recovered inside the monophyletic Physalaemus: (i) the highly supported Physalaemus signifer Clade, which included P. nattereri and the species previously placed in the P. deimaticus and P. signifer Groups; and (ii) the Physalaemus cuvieri Clade, which included the remaining species of Physalaemus. Five species groups were recognized in the P. cuvieri Clade: the P. biligonigerus Group, the P. cuvieri Group, the P. henselii Group, the P. gracilis Group and the P. olfersii Group. The P. gracilis Species Group was the same as that previously proposed by Nascimento et al. (2005). The P. henselii Group includes P. fernandezae and P. henselii, and was the sister group of a clade that comprised the remaining species of the P. cuvieri Clade. The P. olfersii Group included P. olfersii, P. soaresi, P. maximus, P. feioi and P. lateristriga. The P. biligonigerus Species Group was composed of P. biligonigerus, P. marmoratus, P. santafecinus and P. riograndensis. The P. cuvieri Group inferred here differed from that recognized by Nascimento et al. (2005) only by the inclusion of P. albifrons and the exclusion of P. cicada. The paraphyly of P. cuvieri with respect to P. ephippifer was inferred in all the analyses. Distinct genetic lineages were recognized among individuals currently identified as P. cuvieri and they were congruent with cytogenetic differences reported previously, supporting the hypothesis of occurrence of formally unnamed species.

Polytypic and polymorphic cytogenetic variations in the widespread anuran Physalaemus cuvieri (Anura, Leiuperidae) with emphasis on nucleolar organizing regions

We investigated the NOR distribution in ten populations of Physalaemus cuvieri from different regions of Brazil and Argentina. A high variability in NOR pattern was observed and provided a useful tool in grouping several populations. The specimens from the state of Tocantins, northern Brazil, could easily be distinguished from all the other analyzed populations, since its karyotype presented NORs in the chromosome pairs 1,3,4 and 10 (and sometimes also in chromosome 5), and several pericentromeric C-bands. A NOR-site in chromosome 9 characterized three populations from the northeastern region of Brazil. Interestingly, the P. cuvieri populations located in opposite extremes of the geographic distribution had, as a fixed condition, the presence of NORs in 8q int and llp. Besides interpopulational divergences, intrapopulational variability was observed in the number of NORs, except for populations from the states of Bahia and Minas Gerais, which are boundary states respectively in the northeastern and southeastern regions of Brazil. In relation to NOR size, interindividual variations occurred in all Brazilian and Argentinean populations. Additionally, intraindividual variability in NOR size was detected in specimens from Minas Gerais. The data presented herein revealed substantial geographic polytypic variation in P. cuvieri and indicated that a taxonomic reexamination of this species is necessary.